#METABOLOMICS WORKBENCH cbeekman_20240117_111621 DATATRACK_ID:4591 STUDY_ID:ST003140 ANALYSIS_ID:AN005153 PROJECT_ID:PR001952 VERSION 1 CREATED_ON March 26, 2024, 1:50 pm #PROJECT PR:PROJECT_TITLE Spatial analysis of murine GI reveals role of small intestinal bile acid PR:PROJECT_TITLE metabolism in amoxicillin-induced dysbiosis PR:PROJECT_SUMMARY Antibiotics cause collateral damage to resident microbes that is associated with PR:PROJECT_SUMMARY various health risks. To-date, studies have largely focused on impacts of PR:PROJECT_SUMMARY antibiotics on large intestinal and fecal microbiota. Here, we employ a GI-wide PR:PROJECT_SUMMARY integrated multiomic approach to reveal that amoxicillin (AMX) treatment reduces PR:PROJECT_SUMMARY overall bacterial abundance, bile salt hydrolase activity and unconjugated bile PR:PROJECT_SUMMARY acids in the small intestine (SI). An accompanying loss of fatty acids and PR:PROJECT_SUMMARY increase in acyl-carnitines in the large intestine corresponded with PR:PROJECT_SUMMARY spatially-distinct expansions of proteobacteria. Parasutterella PR:PROJECT_SUMMARY excrementihominis utilized fatty acid biosynthesis, becoming dominant in the SI PR:PROJECT_SUMMARY while multiple Klebsiella species employed fatty acid oxidation during expansion PR:PROJECT_SUMMARY in the large intestine. Depletion of bile acids and lipids may contribute to PR:PROJECT_SUMMARY AMX-induced dysbiosis in the lower GI. To test this, we demonstrate that PR:PROJECT_SUMMARY restoration of unconjugated bile acids can mitigate losses of commensals in the PR:PROJECT_SUMMARY large intestine while also inhibiting the expansion of Proteobacteria during AMX PR:PROJECT_SUMMARY treatment. PR:INSTITUTE Brown University PR:DEPARTMENT MMI PR:LABORATORY MMI, Belenky Lab PR:LAST_NAME Beekman PR:FIRST_NAME Chapman PR:ADDRESS BMC 613, 171 Meeting Street, Providence RI 02912 PR:EMAIL Chapman_Beekman@brown.edu PR:PHONE 4012071832 #STUDY ST:STUDY_TITLE Untargeted metabolomic analysis of lumenal contents from AMX-treated and ST:STUDY_TITLE untreated mice ST:STUDY_SUMMARY Antibiotics cause collateral damage to resident microbes that is associated with ST:STUDY_SUMMARY various health risks. To-date, studies have largely focused on impacts of ST:STUDY_SUMMARY antibiotics on large intestinal and fecal microbiota. Here, we employ a GI-wide ST:STUDY_SUMMARY integrated multiomic approach to reveal that amoxicillin (AMX) treatment reduces ST:STUDY_SUMMARY overall bacterial abundance, bile salt hydrolase activity and unconjugated bile ST:STUDY_SUMMARY acids in the small intestine (SI). An accompanying loss of fatty acids and ST:STUDY_SUMMARY increase in acyl-carnitines in the large intestine corresponded with ST:STUDY_SUMMARY spatially-distinct expansions of proteobacteria. Parasutterella ST:STUDY_SUMMARY excrementihominis utilized fatty acid biosynthesis, becoming dominant in the SI ST:STUDY_SUMMARY while multiple Klebsiella species employed fatty acid oxidation during expansion ST:STUDY_SUMMARY in the large intestine. Depletion of bile acids and lipids may contribute to ST:STUDY_SUMMARY AMX-induced dysbiosis in the lower GI. To test this, we demonstrate that ST:STUDY_SUMMARY restoration of unconjugated bile acids can mitigate losses of commensals in the ST:STUDY_SUMMARY large intestine while also inhibiting the expansion of Proteobacteria during AMX ST:STUDY_SUMMARY treatment. ST:INSTITUTE Brown University ST:LAST_NAME Beekman ST:FIRST_NAME Chapman ST:ADDRESS BMC 613, 171 Meeting Street, Providence RI 02912 ST:EMAIL Chapman_Beekman@brown.edu ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 12 ST:NUM_FEMALES 12 ST:PHONE 4012071832 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57/BL6 SU:ANIMAL_ANIMAL_SUPPLIER JAX #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Mouse1 CB1 Treatment:Amox | Sample source:Stomach plate_nr=SPB83Z; position=A1 SUBJECT_SAMPLE_FACTORS Mouse1 CB2 Treatment:Amox | Sample source:PSI plate_nr=SPB83Z; position=A2 SUBJECT_SAMPLE_FACTORS Mouse1 CB3 Treatment:Amox | Sample source:MSI plate_nr=SPB83Z; position=A3 SUBJECT_SAMPLE_FACTORS Mouse1 CB4 Treatment:Amox | Sample source:DSI plate_nr=SPB83Z; position=A4 SUBJECT_SAMPLE_FACTORS Mouse1 CB5 Treatment:Amox | Sample source:Cecum plate_nr=SPB83Z; position=A5 SUBJECT_SAMPLE_FACTORS Mouse1 CB6 Treatment:Amox | Sample source:Colon plate_nr=SPB83Z; position=A6 SUBJECT_SAMPLE_FACTORS Mouse2 CB7 Treatment:Amox | Sample source:Stomach plate_nr=SPB83Z; position=A7 SUBJECT_SAMPLE_FACTORS Mouse2 CB8 Treatment:Amox | Sample source:PSI plate_nr=SPB83Z; position=A8 SUBJECT_SAMPLE_FACTORS Mouse2 CB9 Treatment:Amox | Sample source:MSI plate_nr=SPB83Z; position=A9 SUBJECT_SAMPLE_FACTORS Mouse2 CB10 Treatment:Amox | Sample source:DSI plate_nr=SPB83Z; position=A10 SUBJECT_SAMPLE_FACTORS Mouse2 CB11 Treatment:Amox | Sample source:Cecum plate_nr=SPB83Z; position=A11 SUBJECT_SAMPLE_FACTORS Mouse2 CB12 Treatment:Amox | Sample source:Colon plate_nr=SPB83Z; position=A12 SUBJECT_SAMPLE_FACTORS Mouse3 CB13 Treatment:Amox | Sample source:Stomach plate_nr=SPB83Z; position=B1 SUBJECT_SAMPLE_FACTORS Mouse3 CB14 Treatment:Amox | Sample source:PSI plate_nr=SPB83Z; position=B2 SUBJECT_SAMPLE_FACTORS Mouse3 CB15 Treatment:Amox | Sample source:MSI plate_nr=SPB83Z; position=B3 SUBJECT_SAMPLE_FACTORS Mouse3 CB16 Treatment:Amox | Sample source:DSI plate_nr=SPB83Z; position=B4 SUBJECT_SAMPLE_FACTORS Mouse3 CB17 Treatment:Amox | Sample source:Cecum plate_nr=SPB83Z; position=B5 SUBJECT_SAMPLE_FACTORS Mouse3 CB18 Treatment:Amox | Sample source:Colon plate_nr=SPB83Z; position=B6 SUBJECT_SAMPLE_FACTORS Mouse4 CB19 Treatment:Control | Sample source:Stomach plate_nr=SPB83Z; position=B7 SUBJECT_SAMPLE_FACTORS Mouse4 CB20 Treatment:Control | Sample source:PSI plate_nr=SPB83Z; position=B8 SUBJECT_SAMPLE_FACTORS Mouse4 CB21 Treatment:Control | Sample source:MSI plate_nr=SPB83Z; position=B9 SUBJECT_SAMPLE_FACTORS Mouse4 CB22 Treatment:Control | Sample source:DSI plate_nr=SPB83Z; position=B10 SUBJECT_SAMPLE_FACTORS Mouse4 CB23 Treatment:Control | Sample source:Cecum plate_nr=SPB83Z; position=B11 SUBJECT_SAMPLE_FACTORS Mouse4 CB24 Treatment:Control | Sample source:Colon plate_nr=SPB83Z; position=B12 SUBJECT_SAMPLE_FACTORS Mouse5 CB25 Treatment:Control | Sample source:Stomach plate_nr=SPB83Z; position=C1 SUBJECT_SAMPLE_FACTORS Mouse5 CB26 Treatment:Control | Sample source:PSI plate_nr=SPB83Z; position=C2 SUBJECT_SAMPLE_FACTORS Mouse5 CB27 Treatment:Control | Sample source:MSI plate_nr=SPB83Z; position=C3 SUBJECT_SAMPLE_FACTORS Mouse5 CB28 Treatment:Control | Sample source:DSI plate_nr=SPB83Z; position=C4 SUBJECT_SAMPLE_FACTORS Mouse5 CB29 Treatment:Control | Sample source:Cecum plate_nr=SPB83Z; position=C5 SUBJECT_SAMPLE_FACTORS Mouse5 CB30 Treatment:Control | Sample source:Colon plate_nr=SPB83Z; position=C6 SUBJECT_SAMPLE_FACTORS Mouse6 CB31 Treatment:Control | Sample source:Stomach plate_nr=SPB83Z; position=C7 SUBJECT_SAMPLE_FACTORS Mouse6 CB32 Treatment:Control | Sample source:PSI plate_nr=SPB83Z; position=C8 SUBJECT_SAMPLE_FACTORS Mouse6 CB33 Treatment:Control | Sample source:MSI plate_nr=SPB83Z; position=C9 SUBJECT_SAMPLE_FACTORS Mouse6 CB34 Treatment:Control | Sample source:DSI plate_nr=SPB83Z; position=C10 SUBJECT_SAMPLE_FACTORS Mouse6 CB35 Treatment:Control | Sample source:Cecum plate_nr=SPB83Z; position=C11 SUBJECT_SAMPLE_FACTORS Mouse6 CB36 Treatment:Control | Sample source:Colon plate_nr=SPB83Z; position=C12 SUBJECT_SAMPLE_FACTORS Mouse7 CB37 Treatment:Amox | Sample source:Stomach plate_nr=SPB83Z; position=D1 SUBJECT_SAMPLE_FACTORS Mouse7 CB38 Treatment:Amox | Sample source:PSI plate_nr=SPB83Z; position=D2 SUBJECT_SAMPLE_FACTORS Mouse7 CB39 Treatment:Amox | Sample source:MSI plate_nr=SPB83Z; position=D3 SUBJECT_SAMPLE_FACTORS Mouse7 CB40 Treatment:Amox | Sample source:DSI plate_nr=SPB83Z; position=D4 SUBJECT_SAMPLE_FACTORS Mouse7 CB41 Treatment:Amox | Sample source:Cecum plate_nr=SPB83Z; position=D5 SUBJECT_SAMPLE_FACTORS Mouse7 CB42 Treatment:Amox | Sample source:Colon plate_nr=SPB83Z; position=D6 SUBJECT_SAMPLE_FACTORS Mouse8 CB43 Treatment:Amox | Sample source:Stomach plate_nr=SPB83Z; position=D7 SUBJECT_SAMPLE_FACTORS Mouse8 CB44 Treatment:Amox | Sample source:PSI plate_nr=SPB83Z; position=D8 SUBJECT_SAMPLE_FACTORS Mouse8 CB45 Treatment:Amox | Sample source:MSI plate_nr=SPB83Z; position=D9 SUBJECT_SAMPLE_FACTORS Mouse8 CB46 Treatment:Amox | Sample source:DSI plate_nr=SPB83Z; position=D10 SUBJECT_SAMPLE_FACTORS Mouse8 CB47 Treatment:Amox | Sample source:Cecum plate_nr=SPB83Z; position=D11 SUBJECT_SAMPLE_FACTORS Mouse8 CB48 Treatment:Amox | Sample source:Colon plate_nr=SPB83Z; position=D12 SUBJECT_SAMPLE_FACTORS Mouse9 CB49 Treatment:Amox | Sample source:Stomach plate_nr=SPB83Z; position=E1 SUBJECT_SAMPLE_FACTORS Mouse9 CB50 Treatment:Amox | Sample source:PSI plate_nr=SPB83Z; position=E2 SUBJECT_SAMPLE_FACTORS Mouse9 CB51 Treatment:Amox | Sample source:MSI plate_nr=SPB83Z; position=E3 SUBJECT_SAMPLE_FACTORS Mouse9 CB52 Treatment:Amox | Sample source:DSI plate_nr=SPB83Z; position=E4 SUBJECT_SAMPLE_FACTORS Mouse9 CB53 Treatment:Amox | Sample source:Cecum plate_nr=SPB83Z; position=E5 SUBJECT_SAMPLE_FACTORS Mouse9 CB54 Treatment:Amox | Sample source:Colon plate_nr=SPB83Z; position=E6 SUBJECT_SAMPLE_FACTORS Mouse10 CB55 Treatment:Control | Sample source:Stomach plate_nr=SPB83Z; position=E7 SUBJECT_SAMPLE_FACTORS Mouse10 CB56 Treatment:Control | Sample source:PSI plate_nr=SPB83Z; position=E8 SUBJECT_SAMPLE_FACTORS Mouse10 CB57 Treatment:Control | Sample source:MSI plate_nr=SPB83Z; position=E9 SUBJECT_SAMPLE_FACTORS Mouse10 CB58 Treatment:Control | Sample source:DSI plate_nr=SPB83Z; position=E10 SUBJECT_SAMPLE_FACTORS Mouse10 CB59 Treatment:Control | Sample source:Cecum plate_nr=SPB83Z; position=E11 SUBJECT_SAMPLE_FACTORS Mouse10 CB60 Treatment:Control | Sample source:Colon plate_nr=SPB83Z; position=E12 SUBJECT_SAMPLE_FACTORS Mouse11 CB61 Treatment:Control | Sample source:Stomach plate_nr=SPB83Z; position=F1 SUBJECT_SAMPLE_FACTORS Mouse11 CB62 Treatment:Control | Sample source:PSI plate_nr=SPB83Z; position=F2 SUBJECT_SAMPLE_FACTORS Mouse11 CB63 Treatment:Control | Sample source:MSI plate_nr=SPB83Z; position=F3 SUBJECT_SAMPLE_FACTORS Mouse11 CB64 Treatment:Control | Sample source:DSI plate_nr=SPB83Z; position=F4 SUBJECT_SAMPLE_FACTORS Mouse11 CB65 Treatment:Control | Sample source:Cecum plate_nr=SPB83Z; position=F5 SUBJECT_SAMPLE_FACTORS Mouse11 CB66 Treatment:Control | Sample source:Colon plate_nr=SPB83Z; position=F6 SUBJECT_SAMPLE_FACTORS Mouse12 CB67 Treatment:Control | Sample source:Stomach plate_nr=SPB83Z; position=F7 SUBJECT_SAMPLE_FACTORS Mouse12 CB68 Treatment:Control | Sample source:PSI plate_nr=SPB83Z; position=F8 SUBJECT_SAMPLE_FACTORS Mouse12 CB69 Treatment:Control | Sample source:MSI plate_nr=SPB83Z; position=F9 SUBJECT_SAMPLE_FACTORS Mouse12 CB70 Treatment:Control | Sample source:DSI plate_nr=SPB83Z; position=F10 SUBJECT_SAMPLE_FACTORS Mouse12 CB71 Treatment:Control | Sample source:Cecum plate_nr=SPB83Z; position=F11 SUBJECT_SAMPLE_FACTORS Mouse12 CB72 Treatment:Control | Sample source:Colon plate_nr=SPB83Z; position=F12 SUBJECT_SAMPLE_FACTORS Mouse1 CB73 Treatment:Amox | Sample source:Blood plate_nr=SPB83Z; position=H1 SUBJECT_SAMPLE_FACTORS Mouse2 CB74 Treatment:Amox | Sample source:Blood plate_nr=SPB83Z; position=H2 SUBJECT_SAMPLE_FACTORS Mouse3 CB75 Treatment:Amox | Sample source:Blood plate_nr=SPB83Z; position=H3 SUBJECT_SAMPLE_FACTORS Mouse4 CB76 Treatment:Control | Sample source:Blood plate_nr=SPB83Z; position=H4 SUBJECT_SAMPLE_FACTORS Mouse5 CB77 Treatment:Control | Sample source:Blood plate_nr=SPB83Z; position=H5 SUBJECT_SAMPLE_FACTORS Mouse6 CB78 Treatment:Control | Sample source:Blood plate_nr=SPB83Z; position=H6 SUBJECT_SAMPLE_FACTORS Mouse7 CB79 Treatment:Amox | Sample source:Blood plate_nr=SPB83Z; position=H7 SUBJECT_SAMPLE_FACTORS Mouse8 CB80 Treatment:Amox | Sample source:Blood plate_nr=SPB83Z; position=H8 SUBJECT_SAMPLE_FACTORS Mouse9 CB81 Treatment:Amox | Sample source:Blood plate_nr=SPB83Z; position=H9 SUBJECT_SAMPLE_FACTORS Mouse10 CB82 Treatment:Control | Sample source:Blood plate_nr=SPB83Z; position=H10 SUBJECT_SAMPLE_FACTORS Mouse11 CB83 Treatment:Control | Sample source:Blood plate_nr=SPB83Z; position=H11 SUBJECT_SAMPLE_FACTORS Mouse12 CB84 Treatment:Control | Sample source:Blood plate_nr=SPB83Z; position=H12 #COLLECTION CO:COLLECTION_SUMMARY Lumenal contents collected from multiple GI sites and immediately flash frozen CO:COLLECTION_SUMMARY in liquid nitrogen CO:SAMPLE_TYPE Intestine #TREATMENT TR:TREATMENT_SUMMARY Mice were treated for 24h with amoxicillin/vehicle in drinking water TR:TREATMENT_COMPOUND Amoxicillin TR:TREATMENT_ROUTE oral TR:ANIMAL_ENDP_EUTHANASIA isoflurane, cervical dislocation #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen lumenal contents were lyophilized and 10mg of dry contents were extracted SP:SAMPLEPREP_SUMMARY twice in 300µL in (2:1) acetone and isopropanol. Samples were maintained on dry SP:SAMPLEPREP_SUMMARY ice during extraction steps. Extracts were diluted 1:10 in a solution of 80% SP:SAMPLEPREP_SUMMARY methanol in water. All solvents were LC-MS grade purchased from Fisher SP:SAMPLEPREP_SUMMARY Scientific #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME Agilent 6550 iFunnel LC-MS Q-TOF mass spectrometer CH:COLUMN_NAME none CH:SOLVENT_A none CH:SOLVENT_B none CH:FLOW_GRADIENT none CH:FLOW_RATE 0.150 mL/min CH:COLUMN_TEMPERATURE na #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME General Metabolics #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS A pooled study sample (pSS) was prepared by combining 5 µL of each sample. The MS:MS_COMMENTS pSS was injected every ten samples during data acquisition. Instrumental MS:MS_COMMENTS Analysis Metabolome profiles of the sample extracts were acquired using MS:MS_COMMENTS flow-injection mass spectrometry. The method described here is adapted from MS:MS_COMMENTS Fuhrer et al 2011. The instrumentation consisted of an Agilent 6550 iFunnel MS:MS_COMMENTS LC-MS Q-TOF mass spectrometer in tandem with an MPS3 autosampler (Gerstel) and MS:MS_COMMENTS an Agilent 1260 Infinity II quaternary pump. The running buffer was 60% MS:MS_COMMENTS isopropanol in water (v/v) with 1 mM ammonium fluoride. Hexakis (1H, 1H, MS:MS_COMMENTS 3H-tetrafluoropropoxy)-phosphazene) (Agilent) and 3-amino-1-propanesulfonic acid MS:MS_COMMENTS (HOT) (Sigma Aldrich) were added to the running buffer to serve as lockmasses. MS:MS_COMMENTS The isocratic flow rate was set to 0.150 mL/min. The instrument was run in 4GHz MS:MS_COMMENTS High Resolution, negative ionization mode. Mass spectra between 50 and 1,000 m/z MS:MS_COMMENTS were collected in profile mode. 5 µL of each sample were injected twice, MS:MS_COMMENTS consecutively, within 0.96 minutes to serve as technical replicates. The pooled MS:MS_COMMENTS study sample was injected periodically throughout the batch. Data Processing & MS:MS_COMMENTS Annotation Raw profile data were centroided, merged, and recalibrated using MS:MS_COMMENTS MATLAB software described by Fuhrer et al (10.1021/ac201267k). MS:MS_RESULTS_FILE ST003140_AN005153_Results.txt UNITS:ion counts Has m/z:Yes Has RT:No RT units:No RT data #END